Effect And Possible Mechanism Of B7-H3RNAi-mediated Gene Silencing Combined With Gemcitabine On The Growth Of Pancreatic Cancer Cell-line Patu8988t

Section1: Design of B7-H3siRNA and optimization of transfectionconditionsObjective: To design siRNA targeted on human B7-H3and optimize transfectionconditions.Methods: Following the principle of RNA intefering target sequence sellection,4potential siRNAs of B7-H3were designed after seraching the web resource and database.By Lipofectamine2000, the FAM-negative-siRNA was transfected to Patu8988t cells, andthe efficiency of transfection was measured by fluorescence microscope after optimizingtransfecting conditions. CCK-8was applied to detect the effect of B7-H3gene silencing onthe cell growth of Patu8988t.Results:4siRNAs targeting on B7-H3were successfully constructed and0.125μLLipofectamine2000combined with5pmol siRNA in the96wells board was taken as thebest transfection conditions.Conclusions: The four siRNA targeted on B7-H3were successfully designed andconstructed. The study on effects of B7-H3siRNA on Patu8988t cells will be performed inthe most optimal transfection conditions. Section2: Effect of B7-H3siRNA-mediated gene silencing on cellgrowth of human pancreatic cancer cell line Patu8988tObjective: To investigate the effect of synthesized B7-H3specific siRNA on the cellgrowth and of B7-H3overexpressing human pancreatic cancer cells. Methods: Human pancreatic cancer cell line Patu8988t were cultured and transfectedwith B7-H3siRNA. The most optimal B7-H3siRNA was selected from4siRNAs.RT-PCR and Western blot were used to detect the expression of B7-H3mRNA andprotein.The method of CCK-8was used to examine the cell growth.Results: The expression of B7-H3mRNA and protein in the B7-H3siRNAtransfected-group decreased remarkably（P<0.01），after transfection. B7-H3siRNA did notimpact on the cell growth during the observation period.Conclusions: Transfection of the specific siRNA reduced the expression of B7-H3,but showed no direct inhibition to the growth in Patu8988t cells. Section3: Effect of B7-H3siRNA-mediated gene silencing combinedwith gemcitabine on the growth of Patu8988t cellsObjective: To investigate the effect of B7-H3RNAi-mediated gene silencing on thegrowth of Patu8988t cells combined with GEM.Methods: The suitable concentration of GEM was choosed as10μmol/L. Four groupswere set. Group B（Blank Control Group）: not treated. Group B+（Blank Control WithGEM Group）: cultured with10μmol/L GEM. Group NC+（Negative Control With GEMGroup）: transfected with negative-siRNA and cultured with10μmol/L GEM. Group4+（experimental group）: transfected with the most optimal B7-H3siRNAand cultured with10μmol/L GEM. Group B, Group B+and Group NC+were control groups. Theproliferation of the cells was calculated by the method of CCK-8, early apoptosis of themwas detected by Annexin-V/PI double staining method.Their expressions of Caspase-3mRNA, Caspase-8mRNA, Caspase-9mRNA, Bcl-2mRNA and Bax mRNA were detectedby RT-PCR,and their activities of Caspase-3, Caspase-8and Caspase-9were detected bycorresponding Caspase Activity Kit.Results: Transfected with the most optimal B7-H3siRNA combinated with GEM, theproliferation of Patu8988t cells in experimental group was much lower than control groups(P<0.01). And early stage apoptosis in experimental group was obviously increasedcompared with control groups (P<0.01). The rate of early stage apoptosis in experimental group was (25.83±0.95)%, while (18.6±0.76)%in Blank Control With GEM Group，(20.30±1.08)%in Negative Control With GEM Group. In experimental group， theexpressions of Caspase-3mRNA, Caspase-8mRNA, Caspase-9mRNA were allupragulated，compared with control groups (P<0.05)，but the expressions of Bcl-2mRNAand Bax mRNA were not apparently changed compared with Negative Control With GEMGroup (P＞0.05). The activities of Caspase-3, Caspase-8and Caspase-9in experimentalgroup were increased with significant difference (P<0.05) compared with control groups.Conclusions：B7-H3RNAi-mediated gene silencing could obviously enhance thesensitivity of GEM in pancreatic cancer Patu8988t cells. Its major mechanism maypromote apoptosis of cells.